1. Field of the Invention
The present invention relates to the use of various fractions of hyaluronic acid as osteoinductive agents, i.e., agents capable of stimulating the growth and differentiation of bone forming cells, and therefore the formation of new bone material itself. The type of activity refers to cells of mammalian origin.
2. Description of Related Art
An intricate network of macromolecules constituting the extracellular matrix, a substantial part of the tissue volume, largely fills the extracellular space. In many tissues, such as connective tissues, it is generally more abundant and completely surrounds the cells on all sides, thus determining the physical properties of the tissue. The extracellular matrix is of considerable importance in regulating many processes of tissue behavior. It has been demonstrated that changes in the state and composition oft he extracellular matrix profoundly influence biosynthetic processes and tissue development.
Glycosaminoglycans (GAGs) are the most plentiful non-fibrous, extracellular macromolecules, and are ubiquitous in all connective tissues. This group of high molecular weight anionic glycoconjugates is present in both the soft and mineralized connective tissues. They are substantially composed of large molecules called "non-collagenous proteins" (NCP) of the extracellular matrix, or "proteoglycans". The first of these terms distinguishes these molecules from the group of fibrous proteins such as collagen, elastin, fibronectin and laminin; the second indicates that they are usually found to be covalently bound to protein.
The biological properties of proteoglycans have been the object of intense research, mainly with regard to the soft tissues. The bone and cement, on the other hand, have been virtually ignored as far as their proteoglycan content is concerned. In fact, there is only one report in existence which describes the proteoglycans of the alveolar bone (R. J. Waddington et al., Connective Tissue Res., 1988: 17, 171).
GAGs are long, unbranched polysaccharide chains composed of repetitive disaccharide units. The type of sugar residues, their bonds, and the number and position of the sulfate groups have led to the identification of four main groups of GAGs: 1) hyaluronic acid, 2) chondroitin solfate and dermatan sulfate, 3) heparan sulfate and heparin, and 4) keratan sulfate. It is important to note that GAGs (with the probable exception of hyaluronic acid) rarely exist in a free state within tissues. They are usually covalently bound to proteins.
With the exception of hyaluronic acid, GAGs contain sulfate groups, which together with carboxy groups, form a molecule with a highly negative charge under physiological conditions.
Hyaluronic acid (HA), also called hyaluronan or hyaluronate, contains up to several thousand sugar residues. It is a relatively simple molecule composed of regular sequences of non-sulfated disaccharide units.
HA is considered capable of facilitating cell migration during morphogenesis and tissue repair. It is found in varying quantities in all tissues and fluids in adult animals, and is particularly abundant in early embryos. Because of its simplicity, HA could represent the earliest evolutionary form of glycosaminoglycan. There is a correlation between HA production and mesenchymal cell movement on the one hand, and between HA distribution and cell differentiation on the other (B. Toole et al., Proc. Nat. Acad. Sci., USA, 1972: 69, 1384). The wound healing process possesses some aspects in common with early events which occur during the embryonic development of many organs, and HA plays an important role in both processes (B. P. Toole (1976) in S. H. Barondes, Ed., Neuronal Recognition, Plenum Press, New York, pp. 275-329); a correlation has been found between HA and cell adhesion, in terms of HA receptors (Goldstein et al., Cell, 1989: 56, 1063; I. Stamenkovic et al, Cell, 1989: 56, 1057).
Most of the characteristics of HA noted above for connective tissue can also be observed in bone. The probability that noncollagenous proteins influence local calcification mechanisms was assessed some time ago (H. Iwata et al., Clin. Orthop., Rel. Res. 1973: 90, 236; M. R. Urist, in Bourne, G. H. ed.: The Biochemistry and physiology of Bone, New York, Academic Press, 1976: 1-59). There are a number of interrelationships between HA and bone formation:
1. HA is a prominent component of the extracellular matrix during morphogenesis of bone (B. Toole et al., Develop. Biol., 1971: 26, 28; H. Iwata et al., Clin. Orthop., Rel. Res., 1973: 90, 236);
2. Considerable quantities of HA are present during the transition of mesenchymal cells to cartilage (O. Wiebkin et al., FEBS Lett., 1973: 37, 42; C. H. Handley et al., Biochem. Biophys. Acta, 1976: 444, 69);
3. In terms of its correlation with wound healing processes and bony tissue development (B. P. Toole (1976) in S. H. Barondes, Ed., Neuronal Recognition, Plenum Press, New York, pp. 275-329), HA can be considered as a sort of "primer".
It has long been known that HA is useful in the treatment of periodontal diseases (Minerva Stomatol. 17: 140, 1968; Riv. Ital., Stomatolog. 20: 1540, 1965). There seems to be well-defined sequence of events in these cases:
--First, a HA-rich matrix is deposited in a space poorly furnished with cells;
--Secondly, cell migration is stimulated and the HA matrix is infiltrated by cells migrating from adjacent tissues; and
--Lastly, the cells inside the extracellular matrix secrete hyaluronidase (which degrades the HA), sulfated glycosaminiglycans, and collagen, which replace part of the HA while the matrix becomes remodelled.
In each of the three developmental systems noted above, the HA matrix is first synthesized and then degraded. Consequently, it is probable that this transitory HA matrix, poor in cells, is needed before the complicated series of cell-mediated events which follows its destruction can proceed.
The presence of a certain number of sites binding HA onto the different types of cell surfaces has also been reported, such as in endothelial cells and fibroblasts (S. Eriksson et al., Exp. Cell Res. 1983: 144, 223; R. H. Raja et al., J. Cell. Biol.; 1985: 101, 426a; C. B. Underhill et al., Cell Biol. 1979: 82, 475). Thus, HA is able to interact specifically both with the cell surfaces and with the molecules of the extracellular matrices.
The chemical and physical-chemical characteristics involved in HA's osteoinductive activity have not yet been identified. It is known that the biological activity of hyaluronic acid can often be associated with definite fractions of different molecular weights and viscosity.